The present invention relates to obtaining antibodies recognizing lipids and more particularly, is related to methods for obtaining antibodies against lipidic structures different from the lipidic bilayer, and to the use of these antibodies in diagnostic and/or treatment of diseases associated with the antiphospholipid syndrome; as well as for the determination of physiological states of the cell.
Considering the state of the art there are different studies in which evidence of the existence of antibodies that recognize lipids can be found. For example, they have been detected in the serum of patients with antiphospholipid syndrome, as was described by Asherson et al. in their book xe2x80x9cThe antiphospholipid syndromexe2x80x9d in 1996 (CRC Press, Boca Raton). In the same way, antiphospholipid antibodies have been obtained from animals that were experimentally treated with lipids by active immunization, in accordance with Alving in 1992 (Biochim. Biophys. Acta 1113:307-322) or, in animals that received antiphospholipid antibodies by passive immunization, as Tincani and Shoenfeld described in 1996 in the above mentioned book.
The anti-lipid antibodies have been classified into two major subgroups according with the method used for their determination. These groups are anti-cardiolipin antibodies and anticoagulant antibodies (Guglielmone y Fernandez, 1998, J. Rheumatol. 26:86-90).
The anti-cardiolipin antibodies are determined by methods in which cardiolipin immobilized in a solid phase is used. This was described by Harris et al. in 1985 (Clin. Rheum. Dis. 11:591-609), such as the enzyme-linked immunosorbent assays and the radioimmunoassays better known by their respective initial abbreviations as ELISA and RIA which have been broadly used in the above mentioned technique.
The anticoagulant antibodies are detected by methods in which the prolongation in the coagulation time of plasma samples is measured in vitro, according with Bevers et al. 1991(Thromb. Haemost. 66:629-632). Some of these methods are: activated partial thromboplastin time (APTT), dilute Russell""s viper venom time (dRVVT), protein C, and protein S among others. In these methods, the anticoagulant antibodies are bound to phosphatidylethanolamine or to phosphatidylserine which are intermediary factors in the blood coagulation cascade, and when the concentration of these lipids decrease due to the immune reaction, the coagulation time is prolongated.
The anti-cardiolipin antibodies have the disadvantage of producing crossed reaction with other anionic lipids such as phosphatidylserine and phosphatidylglycerol. Due to the lack of specificity for a certain type of lipid, the above mentioned antibodies are generally known as antiphospholipid antibodies.
In addition, antibodies against phosphatidylethanolamine have been detected in the sera from patients with antiphospholipid syndrome. Also, antibodies against phosphatidylcholine are detected in patients with hemolytic anemia, as was described by Sugi and McIntyre (Blood 86:3083-3089) and Arvieux et al. (Thromb. Haemost. 74:1120-1125), respectively, in 1995.
On the other hand, some studies have demonstrated that the binding of antiphospholipid antibodies to the lipidic antigen increases in the presence of a plasmatic protein. For example, in 1990, McNeil et al., determined that the binding of antibodies to the cardiolipin was markedly enhanced by the plasma protein xcex22-glycoprotein I or apoprotein H (Proc. Nat. Acad. Sci. USA 87:4120-4124). Additionally, some anti-cardiolipin antibodies are bound directly to xcex22-glycoprotein I, as was described by Roubey et al. in 1995 (J. Immunol. 154:954-960). These findings suggest that the anti-cardiolipin antibodies may recognize either a cryptic epitope on xcex22-glycoprotein I exposed on the complex of xcex22-glycoprotein I-cardiolipin, or xcex22-glycoprotein I alone but with a very low affinity towards he glycoprotein, as was described by Pengo et. al. (1995, Thromb. Haemost. 73:29-34).
In accordance with these studies, it may be concluded that the binding of antiphospholipid antibodies to lipidic antigens is also associated with proteins. Sugi and McIntyre (op. cit., 1995) found that the proteins called kininogens are involved in the binding of antibodies to phosphatidylethanolamine, whereas the proteins that are bound to phosphatidylserine, such as prothrombin, protein C, protein S and annexin V, have been implicated in the binding of anticoagulant antibodies to phosphatidylserine, according with the studies in 1994 by Nakamura et al. (Biochim. Biophys. Res. Commun. 205:1488-1493) and by Roubey (Blood 84:2854-2867).
These studies indicate that the antigen of some antiphospholipid antibodies is really a complex formed by phospholipids and specific plasma proteins, but these proteins differ from those required for reactivity of antiphospholipid antibodies with cardiolipin. Nevertheless, in other studies, antiphospholipid antibodies that bound directly to the phospholipid have been identificated such as the anti-cardiolipin antibodies that do not require the xcex22-glycoprotein I. Such studies were carried out by McNeil et al. in 1989 (Br. J. Haematol. 73:506-513) and by Pengo and Basiolo in 1993 (Thromb. Res. 72:423-430).
On the other hand, some anti-cardiolipin antibodies, purified by affinity chromatography, do not show anticoagulant activity (McNeil et al., op. cit., 1989; Shi et al., 1993, Blood 81:1255-1262). However, other studies demonstrated that the anti-cardiolipin and the anticoagulant antibodies were removed by adsorption with cardiolipin (Pengo and Biasiolo, op. cit., 1993; Pierangeli et al. 1993, Br. J. Haematol. 85:124-132).
Additionally, during studies in experimental animals, treated by passive or active immunization, the employed methods for the detection of antiphospholipid antibodies are the same as those described for the detection of human antiphospholipid antibodies. Furthermore, in these animal models, the different organs and tissues were analized by anatomical and histopathological studies, by immunofluorescent studies, and even by fetal resorption analysis and consequently the produced lessions in fetuses and placentas of the female animal models were also analyzed. These works were performed by Tincani y Shoenfeld (op. cit. 1996) and by Shoenfeld and Ziporen (Lupus 7:S158-S161, 1998).
The previously mentioned studies, show that the antiphospholipid antibodies described in human patients and in animal models have a broad specificity towards the lipidic antigens. This broad specificity of the antibodies may be attributed, among other causes, to the lack of specificity of the methods used for the detection of the above described antibodies.
In such methods, it has not been considered the chemical structure and the molecular association of lipidic antigens, as well as the chemical properties that the lipidic antigens have in the nature. As a consequence, in the lipidic antigens that have been used in those methods, the phospholipids are bound to artificial solid supports, such as in the ELISA and RIA methods, or they are in a molecular association that is not completely characterized, like in tests where the prolongation in the coagulation time is detected.
There are only a few studies in which the molecular structure of the phospholipid employed as antigen has been considered. For example, the reports of Rauch et al. in 1989 and in 1998 (Thromb. Haemost. 62:892-896 and Thromb. Haemost. 80:936-941, respectively) and that of Berard et al. (J. Lab. Clin. Med., 1993, 122:601-605). In these reports, the authors demonstrated that the sera from some patients with systemic lupus erythematosus is inhibited in its anticoagulant activity by phosphatidylethanolamine associated in the hexagonal tubular II phase. This inhibition was not observed when the phospholipid was associated into the bilayer phase. However, the properties of the cellular membrane can not be related with the tubular association of phospholipids because this tubular lipidic association is practically incompatible with the vesicular structure of the cellular membrane, as different authors have established. In other words, in the lipidic antigens used in these studies the phospholipids are in molecular arrangements that do not correspond to the molecular arrangements that they present in the cellular membrane.
Additionally, it is well-known that the molecular structure of the plasmatic membrane of mammal cells is like an association heteropolymer formed by phospholipids, glycolipids, cholesterol, proteins and glycoproteins where the lipids are mainly in a molecular arrangement of bilayer. Nevertheless, it is also known that lipids may have molecular arrangements different to the bilayer and that such arrangements depend on the molecular geometry of the lipids and the surrounding conditions.
Cylindrical shaped lipids, such as phosphatidate, phosphatidylglycerol, phosphatidylinositol, phosphatidylcholine, phosphatidylserine, cardiolipin, sphingomyelin and diglucosyldiacylglycerides, in an aqueous media are associated in closed bilayers, or liposomes. Cylindrical lipids constitute from 60 to 70% of the membranal lipids.
On the other hand, the conic shaped lipids such as phosphatidylethanolamine, monoglucosyldiacylglycerides, and diacylglycerols, as well as the above mentioned lipids: phosphatidate, cardiolipin, phosphatidylserine, and phosphatidylglycerol but in the presence of divalent cations are assembled in the molecular phase known as hexagonal II (HII), which corresponds to tubular cylinders packed hexagonally. While the inverted cone shaped lipids, such as lysophospholipids and gangliosides are associated in micelles. Conic and inverted conic shaped lipids represent from 30 to 40% of the membranal lipids.
Lipidic arrangements in hexagonal II or micellar phases, as well as any other structural arrangement of lipids that do not form a bilayer but that is immerse in a bilayer, are considered, for the purposes of this invention, as lipidic structures different to the lipidic bilayer or xe2x80x9clipidic particlesxe2x80x9d, independently of the kind of lipids that are forming these structures.
In the same way, it is known that in the presence of divalent cations, drugs like chlorpromazine and procainamide, non-polar peptides, proteins such as the protein of the bacteriophage M13, cholesterol, lanthanum ions, as well as changes in temperature and in the pH, the conic lipids form molecular arrangements different to the lipidic bilayer. These lipidic arrangements are of transient nature because when the concentration of the compounds that induced their formation diminishes or when the temperature or the pH changes again, the conic shaped lipids return to the bilayer arrangement as was described by Cullis et al., in 1991 (Membrane Fusion. Marcel Dekker, New York), by Baeza et al. in 1995 (Biochem. Cell Biol. 73:289-297) and Aguilar et al., in 1999 (J. Biol. Chem. 274:25193-25196). Lipidic bilayer molecular arrangements are observed like a smooth surface by cryofracture analysis.
Lipids in general are molecules with low immunogenicity, and of the two molecular arrangements that the lipids may adopt in cellular membranes, it is considered that the lipidic bilayer will be the less immunogenic because it is the one that mainly constitutes the matrix of all cellular membranes.
However, it is known that the lipidic structures different to the bilayer, which are stabilized with divalent cations and that are observed as protuberances on the smooth surface of the bilayer by cryofracture analysis, induce the formation of antibodies that recognize the lipids that are associated in lipidic particles and they do not react with lipids associated in bilayer.
In connection with the above-mentioned studies, Baeza and their collaborators in 1995 (op. cit.) reported the elaboration of liposomes with lipid molecular arrangements different to the bilayer, as well as the antigenic activity of these molecular arrangements, because they were able to obtain polyclonal antibodies with them. By means of cytofluorometric analysis of the immune reaction they were also able to identify the presence of lipidic structures in the liposomes described, using for it anti-lipidic particles polyclonal antibodies obtained from mice sera.
To this respect, the mice were immunized by the introduction of artificially formed lipidic particles which when are present in excess caused the wanted immune reaction. Until now, it is believed that molecular arrangements different to the bilayer or lipidic particles would be also scarce immunogenics when they are present in the nature, for example in cells of human and animals, because lipidic particles are transient and therefore they would not be detected by immune systems.
Additionally, from the analysis of the above mentioned studies, one can observe that the cardiolipin is the only lipid that has been able to react with antibodies present in patients with the antiphospholipid syndrome or associated illnesses, and that the other phospholipids usually present in the cellular membrane in general require to be associated with proteins to react with the antibodies from these patients, or, they require to be associate in a molecular arrangement incompatible with the molecular structure of the cellular membrane; with the exception of the studies of Baeza and their collaborators (op. cit. 1995) on the anti-lipidic particles antibodies which react with a lipidic molecular arrangement similar to the one that has been described in cellular membranes.
To this respect, the presence in sera from patients with the antiphospholipids syndrome of anti-cardiolipin antibodies, a mitochondrial lipid, of anti-nuclear antibodies and of anti-DNA antibodies, it is indicative of the existence of previous events that cause immunologic damage to cellular membranes, with the disruption of the cells and the exhibition of the intracellular components to the immunologic system, causing the corresponding immunologic reaction that contributes to the development of the syndrome. However, up to now there have not been found studies which allow to determine the events that cause the disruption of the cellular membrane. In other words, with the existent knowledge so far it is impossible to detect the anti-cardiolipin antibodies, the anti-nuclear or even the anti-DNA antibodies before the damage that has been caused to the cell, impeding an early diagnosis and treatment of the illnesses associated with the syndrome.
Additionally, in the Doctoral Thesis presented by Leopoldo Aguilar in Dec. 17, 1997 (Determination of non-bilayer lipidic arrangements in liposomes and cellular membranes with monoclonal antibodiesxe2x80x9d, Doctoral Thesis, National School of Biological Sciences, National Polytechnic Institute, Mxc3xa9xico) 5 sera from patients with primary antiphospholipid syndrome and 5 sera from patients with systemic lupus erythematosus were analyzed, these illnesses were corroborated by clinical characteristics that the patients presented and by means of the detection of anti-cardiolipin antibodies, and of anti-nuclear antibodies, these last ones in the case of the sick persons with lupus. The analyzed sera from all patients also presented anti-lipidic particles antibodies, detected according to the techniques of liposomal-ELISA and of liposomal cytofluorometry described in the above mentioned Thesis.
This discovery, however, does not show any advantage for the early detection of the illnesses, since the presence of the antiphospholipid antibodies and of the anti-lipidic particles antibodies in those patients can be explained according with two hypothesis.
The first one, assumes that an unknown factor causes the destruction of the cellular membrane, which promotes the formation of lipidic particles from the membranal lipids that enter in contact with the immunologic system together with the intracellular components, with the consequent simultaneous formation of anti-lipidic particles antibodies, and anti-cardiolipin and anti-nuclear antibodies.
The second hypothesis, consists on assuming that the lipidic particles are formed in the cellular membrane before its destruction, and they would form anti-lipidic particles antibodies that would destroy the membrane, exposing the intracellular components to the immunologic system and giving place later on to the formation of anti-cardiolipin and anti-nuclear antibodies.
This second hypothesis was proposed in the Master Thesis presented by Monica Lara on Aug. 20, 1999 (xe2x80x9cDetection of anti-lipidic particles antibodies in patients with the anti-phospholipid syndrome,xe2x80x9d Master Thesis, Escuela Nacional de Ciencias Biolxc3x3gicas [National School of Biological Sciences], Instituto Politxc3xa9cnico Nacional [National Polytechnic Institute], Mexico.
So far, none of the two hypothesis has been demonstrated, which is of supreme importance for the treatment of the illnesses, since should the second hypothesis probed to be certain, it would be possible to detect the illnesses above mentioned in their early stages, and also, it would be possible the prevention, cure or patient""s improvement from such illnesses.
Derived from the above-mentioned hypothesis, it has been aimed to suppress the inconveniences of the induction and detection of antiphospholipid antibodies techniques caused by the structure and molecular association of the antigens used in these methods, by the employment of lipidic antigens with a structure and molecular association similar to the one found in patients with illnesses associated with antiphospholipids antibodies. These novel lipidic antigens have been used for the induction and detection of anti-lipidic particles antibodies that allow an early diagnosis of these illnesses, as well as for the determination of physiologic states of the cell, as apoptosis, or programmed cellular death (Pittoni and Isenberg, 1998, Semin. Arthritis. Rheum. 28:163-178) and those which are present in the cellular cycle (Go, G1, G2 and M) among others.
Keeping in mind the deficiencies in the structure and in the molecular association of the antigens that are used in the techniques of induction and detection of antiphospholipid antibodies from the methods of the previous techniques, one of the objectives of the present invention consists on using lipidic antigens with a structure and molecular association similar to the one that is present in patients with illnesses associated with antiphospholipid antibodies, with the purpose of providing a method for the detection of anti-lipidic particles antibodies.
It is another objective of the present invention, to provide a diagnosis method which uses monoclonal antibodies specific to lipidic antigens that respond in the same way that the anti-lipidic particles antibodies present in sera from patients with diverse illnesses associated with antiphospholipid antibodies, with the purpose of designing a strategy for the treatment of these patients against such illnesses.
It is an additional objective of the present invention, to provide a kit or diagnosis set for the detection of anti-lipidic particles antibodies in early stages of illnesses that present such antibodies in animals and in humans.
It is another objective of the present invention, to provide a kit or diagnosis set for th detection of lipidic particles in the membranes of the cells of ill entities, human or animal, that present anti-lipidic particles antibodies.
It is still another objective of the present invention, to provide a method for the prevention, cure or patient""s improvement from such illnesses by means of the inhibition or the blockage of anti-lipidic particles antibodies.
Another objective of the present invention still consists on providing a method for the prevention, cure or patient""s improvement from such illnesses, by means of the stabilization of cellular membranes that impedes the formation of lipidic particles and therefore the later formation of anti-lipidic particles antibodies.
An additional objective of the present invention consists on providing methods and its corresponding kits for the detection of the different physiologic states that can present the cells, which can lead to the prevention of illnesses related with antiphospholipid antibodies.